Process for the fermentative production of cephalosporin

ABSTRACT

A method for the recovery of a compound formula (I) from a complex mixture,                    
     comprising the steps of: (a) acidifying the complex mixture to a pH below 6.5 and maintaining the mixture below said pH at a temperature of between 50° C. and 130° C.; and/or (b) contacting the complex mixture with a carbon dioxide source; and (c) subjecting the mixture obtained after steps (a) and/or (b) to chromatography to obtain the compound of formula (I).

FIELD OF THE INVENTION

The present invention relates to a process for the preparation ofcephalosporins and cephalosporin derivatives. More in particular, thepresent invention relates to the recovery of cephalosporins andderivatives thereof from complex mixtures of cephalosporins and otherbeta-lactam compounds. The invention is also concerned with recovery ofdeacylated cephalosporins from mixtures of beta-lactam compounds andside chains, such as hose obtainable by enzymatic side-chain removal.

BACKGROUND OF THE INVENTION

Semi-synthetic routes to prepare cephalosporins mostly start fromfermentation products such as penicillin G, penicillin V andCephalosporin C, which are converted to the corresponding β-lactamnuclei, for instance in a manner as is disclosed in K. Matsumoto,Bioprocess. Techn., 16, (1993), 67-88, J. G. Shewale & H. Sivaraman,Process Biochemistry, August 1989, 146-154, T. A. Savidge, Biotechnologyof Industrial Antibiotics (Ed. E. J. Vandamme) Marcel Dekker, New York,1984, or J. G. Shewale et al., Process Biochemistry International, June1990, 97-103. The obtained β-lactam nuclei are subsequently converted tothe desired antibiotic by coupling to a suitable side chain, as has beendescribed in inter alia ED 0 339 751, JP-A-53005185 and CH-A-640 240. Bymaking different combinations of side chains and β-lactam nuclei, avariety of penicillin and cephalosporin antibiotics may be obtained.

7-Amino desacetoxy cephalosporanic acid (7-ADCA) and7-aminocephalosporonic acid (7-ACA) are known to be the most importantintermediates for the production of antibiotics used in thepharmaceutical industry. 7-ADCA is for example obtained by chemical orenzymatic cleavage (deacylation) of phenylacetyldesacetoxycephalosporanic acid yielding 7-amino desacetoxy cephalosporanic acidand phenyl acetic acid.

Phenylacetyldesacetoxy cephalosporanic acid is normally produced bychemical treatment of penicillin G sulfoxide, which is formed frompenicillin G. In this production process a large amount of chemicals arerequired to ensure that the desired reaction take place. This is bothexpensive and places a heavy burden on waste management. Moreover, thetotal yield of the process, is not very high.

To overcome some of the drawbacks of the chemical process a fermentativeprocess has been disclosed for the production of 7-ADCA, 7-aminodesacetyl cephalosporanic acid (7-ADAC) and 7-ACA, involvingfermentative production of N-substituted β-lactams, such asadipyl-7-ADCA, adipyl-7-ADAC or adipyl-7-ACA by a recombinantPenicillium chzysogenum strain capable of expressing adesacetoxycephalosporanic acid synthetase (DAOCS) also known as“expandase” from a transgene (EP 0 532 341, EP 0 540 210, WO 93/08287,WO 95/04148). The expandase takes care of the expansion of the5-membered ring of certain N-acylated penicillanic acids, therebyyielding the corresponding N-acylated desacetoxycephalosporanic acids.

In order to yield the economically most important non-acylatedcephalosporins, such as 7-ADCA, 7-ADAC and 7-ACA, the acyl groups areenzymatically removed with a suitable acylase.

Known processes for recovering chemically or enzymatically producedpenicillanic and cephalosporanic acids are not effective for therecovery of the N-substituted β-lactam intermediates and deacylatedamino-β-lactams. The main problem with the recovery of thefermentatively produced cephalosporin compounds mentioned above is thecomplexity of the broth, or culture filtrate. The broth usuallycomprises various penicillanic acids, such asalpha-aminoadipyl-6-penicillanic acid,alpha-hydroxyadipyl-6-penicillanic acid, 6-aminopenicillanic acid(6-APA), various cephalosporanic acids including alpha-aminoadipyl- andhydroxyadipyl-7-ADCA and a lot of proteinaceous material. Known recoveryprocedures do not give an acceptable quality of the cephalosporanic acidproduct in terms of purity. In deacylation this leads to problems interms of reduced enzyme half-life, slower bioconversion rate and moreexpenses in the recovery after bioconversion and/or unacceptablecontaminant levels. Moreover, after deacylation, such impurities preventor at least hamper the recovery of the desired deacylated cephalosporincompound of the desired specifications.

SUMMARY OF THE INVENTION

The invention provides for a method for the recovery of acephalosporanic acid compound of the general formula (I):

wherein

R₀ is hydrogen or C₁₋₃ alkoxy;

Y is CH₂, oxygen, sulphur, or an oxidised form of sulphur;

R₁ is any of the groups selected from the group consisting of

hydrogen,

hydroxy,

halogen,

saturated or unsaturated, straight or branched alkyl (1-5 carbon atoms;optionally replaced by one or more heteroatoms), optionally substitutedwith hydroxy, halogen, aryl, alkoxy (1-3 carbon atoms), or acyl;

alkoxy (1-3 carbon atoms; optionally replaced by one or moreheteroatoms), optionally substituted with hydroxy or halogen; or

cycloalkyl (3-8 carbon atoms) optionally substituted with hydroxy,halogen, amino;

aryl;

heteroaryl; and

R₂ is selected from the group consisting of adipyl(1,4-dicarboxybutane), succinyl, glutaryl, adipyl, pimelyl, surberyl,2-(carboxyethylthio)acetyl, 3-(carboxy-ethylthio)propionyl, higher alkylsaturated and higher alkyl unsaturated dicarboxylic acids,

from a complex mixture comprising in addition to the compound of thegeneral formula 6-aminopenicillanic acid (6-APA) and optionally one ormore N-substituted β-lactam compounds,

comprising the steps of:

(a) acidifying the complex mixture to a pH below 6.5 and maintaining themixture below said pH at a temperature of between 10° C. and 150° C.;and/or

(b) contacting the complex mixture with a carbon dioxide source; and

(c) recovering the cephalosporanic acid compound of the formula (I) fromthe mixture obtained after steps (a) and/or (b).

Preferably in step (a) the temperature is kept between about 50° C. andabout 130° C., preferably between 70 and 120° C., for between 10 secondsand about 1 week and the pH is kept at or below pH 4.5. According to apreferred method the compound of formula (I) has been produced byfermentation of a micro-organism capable thereof, the complex mixturebeing a broth, a culture filtrate or any culture liquid derivable fromthe broth after fermentation.

Preferred compounds of the general formula (I) are selected from thegroup consisting of adipyl-7-ADCA, adipyl-7-ADAC and adipyl-7-ACA.

According to another aspect of the invention step (c) is performed bysubjecting the mixture obtained after steps (a) and/or (b) tochromatography, preferably adsorption chromatography, more preferablyHydrophobic Interaction Chromatography.

According to another aspect of the invention the use of chromatographyin a process or recovering a cephalosporin compound according to formula(I) is provided, preferably by adsorption chromatography, morepreferably Hydrophobic Interaction Chromatography, still more preferablyusing Simulated Moving Bed technology.

According to yet another aspect of the invention a method is providedfor making a compound of formula (II):

wherein

R₀ is hydrogen or C₁₋₃ alkoxy;

Y is CH₂, oxygen, sulphur, or an oxidised form of sulphur;

R₁ is any of the groups selected from the group consisting of

hydrogen,

hydroxy,

halogen,

saturated or unsaturated, straight or branched alkyl (1-5 carbon atoms;optionally replaced by one or more heteroatoms), optionally substitutedwith hydroxy, halogen, aryl, alkoxy (1-3 carbon atoms), or acyl;

alkoxy (1-3 carbon atoms; optionally replaced by one or moreheteroatoms), optionally substituted with hydroxy or halogen; or

cycloalkyl (3-8 carbon atoms) optionally substituted with hydroxy,halogen, amino; aryl;

heteroaryl;

comprising the steps of making a compound according to formula (I)wherein R₀, Y and R₁ are as above and R₂ is selected from the groupconsisting of adipyl (1,4-dicarboxybutane), succinyl, glutaryl, adipyl,pimelyl, surberyl, 2-(carboxyethylthio)acetyl,3-(carboxyethylthio)-propionyl, higher alkyl saturated and higher alkylunsaturated dicarboxylic acids; deacylating the compound of formula (I)to obtain a conversion solution which comprises a compound according toformula (II).

The conversion solution preferably further comprises the cleaved sidechain designated R₂.

According to a preferred embodiment, the process comprises the furtherstep of recovering the compound of formula (II) from the solution bycrystallisation, preferably preceded and/or followed (aftersolubilisation of the crude crystals i.e. by crystallisation) bytreatment of the solution with an agent selected such as activatedcarbon or an adsorber resin. According to another aspect of theinvention during or before crystallisation and/or recrystallisation asolvent such as methanol, ethanol, (iso)propanol, isobutanol, n-butanol,or acetone or a combination of any of the mentioned agents is added.Preferred adsorber resins are selected from XAD16 (CAS No. 102419-63-8),XAD1600 (CAS No. 153796-66-8) and HP20 (CAS No. 55353-13-4). Preferredaccording to the invention is a method wherein the 6-aminopenicillanicacid (6-APA) level is 10 ppm or less with respect to the compound offormula (II). According to another aspect, a process is provided whereinfollowing the deacylation the solution is treated to remove, at leastpartially, the cleaved side chain represented by R₂. This step may beperformed, or repeated, after crystallisation and solubilisation (i.e.recrystallisation) of the compound of formula (II). Also removal of thecleaved side-chain may be carried out on the mother liquor obtainedafter crystallisation or recrystallisation.

Thus, a process is provided wherein said treatment to remove, at leastpartially, the cleaved side chain is followed by solubilisation of thecrude crystals and recrystallisation of the compound of formula (II).

Preferably said treatment to remove the cleaved side chain comprisessubjecting the conversion solution, or the mother liquor, or both, tomembrane filtration a pH below 5, preferably below 4, more preferablynear or below 3. Accordingly, the use is provided of membrane filtrationto remove a dicarboxylic acid from a mixture comprising the dicarboxylicacid and a β-lactam antibiotic. The mixture is preferably a motherliquid obtained after crystallisation of a compound of the formula (II)or the mixture obtained after deacylation of the compound of formula(I). Membrane filtration takes preferably place at a pH of about 5 orless, preferably at pH 4 or less, yet more preferably by nanofiltrationat or below pH 3.

According to another aspect of the invention, a process is providedwherein the side chain R₂ is, at least partially, removed from theconversion mixture by crystallisation and/or recrystallisation.

According to still another aspect of the invention, a process isprovided wherein the side chain R₂ is, at least partially, removed fromthe conversion mixture by acidifying the mixture to a pH lower than 3and next contacting this mixture with an organic solvent, for instanceamyl acetate, butyl acetate, ethyl acetate, methyl isobutyl ketone,cyclohexanone, isobutanol or n-butanol.

DETAILED DESCRIPTION OF THE INVENTION

The invention pertains to a method for the recovery of a cephalosporanicacid compound of the general formula (I):

wherein

R₀ is hydrogen or C₁₋₃ alkoxy;

Y is CH₂, oxygen, sulphur, or an oxidised form of sulphur;

R₁ is any of the groups selected from the group consisting of

hydrogen,

hydroxy,

halogen,

saturated or unsaturated, straight or branched alkyl (1-5 carbon atoms;optionally replaced by one or more heteroatoms), optionally substitutedwith hydroxy, halogen, aryl, alkoxy (1-3 carbon atoms), or acyl;

alkoxy (1-3 carbon atoms; optionally replaced by one or moreheteroatoms), optionally substituted with hydroxy or halogen; or

cycloalkyl (3-8 carbon atoms) optionally substituted with hydroxy,halogen, amino;

aryl;

heteroaryl; and

R₂ is selected from the group consisting of adipyl(1,4-dicarboxybutane), succinyl, glutaryl, adipyl, pimelyl, surberyl,2-(carboxyethylthio)acetyl, 3-(carboxy-ethylthio)propionyl, higher alkylsaturated and higher alkyl unsaturated dicarboxylic acids,

from a complex mixture comprising in addition to the compound of thegeneral formula 6-aminopenicillanic acid (6-APA) and optionally one ormore N-substituted penicillanic acid compounds,

comprising the steps of:

(a) acidifying the complex mixture to a pH below 6.5 and maintaining themixture below said pH at a temperature of between 10° C. and 150° C.;and/or

(b) contacting the complex mixture with a carbon dioxide source; and

(c) recovering the cephalosnoranic acid compound of the formula from themixture obtained after steps (a) and/or

(b). The invention relates further to a process for the preparation ofcephalosporins having the general formula (II):

wherein

R₀ is hydrogen or C₁₋₃ alkoxy;

Y is CH₂, oxygen, sulphur, or an oxidised form of sulphur;

R₁ is any of the groups selected from the group consisting of

hydrogen,

hydroxy,

halogen,

saturated or unsaturated, straight or branched alkyl (1-5 carbon atoms;optionally replaced by one or more heteroatoms), optionally substitutedwith hydroxy, halogen, aryl, alkoxy (1-3 carbon atoms), or acyl;

alkoxy (1-3 carbon atoms; optionally replaced by one or moreheteroatoms), optionally substituted with hydroxy or halogen; or

cycloalkyl (3-8 carbon atoms) optionally substituted with hydroxy,halogen, amino;

aryl;

heteroaryl.

The compound according to formula (I) may be produced by any series ofsteps which yield a complex mixture as defined herein, from which therecovery of the compound according to formula (I) is accomplished. Forthe purposes of the specification and claims, a complex mixture isdefined as a mixture comprising a N-substituted cephalosporin compoundand substituted or unsubstituted β-lactam compounds.

The compound of formula (II) is obtained by the following series ofsteps:

(a) recovering, preferably purifying, the compound of formula (I);

(b) deacylating the preferably purified compound of formula (I) toobtain a solution comprising the compound of formula (II) (theconversion solution); and

(c) recovering, preferably purifying the compound of formula (II).

One of the obstacles of producing N-substituted cephalosporanic acidfermentatively is the presence of unwanted contaminating β-lactamcomponents, for instance N-substituted 6-amino penicillanic acid.According to one embodiment of the invention, it has been found thatthese contaminations can be remarkably reduced by incubating the broth,the filtrate of the broth or a liquid derived from the broth using anybiomass separation technique, under acidified conditions, preferablyaccompanied with an elevated temperature. The broth is acidified down toa pH lower than 6.5, preferable lower than 4.5, using at least one knownacid, for instance sulphuric acid, hydrochloric acid or nitric acid or acombination thereof. Operating temperature is in the range of 20 to 150°C., preferably at 70 to 120° C. Residence time at these conditions is inthe range of a few seconds (at 150° C.), or several days (at 20° C.),preferably 10 seconds to 60 minutes. The pH/temperature treatment ispreferably carried out for a period which provides for an N-substituted6-APA reduction of a factor 100, preferably 1000, more preferably1,000,000 with respect to the compound of formula (II). This seep can becarried out either before or after biomass separation and can beperformed batch wise or continuously.

According to another embodiment of the invention contaminatingpenicillin components, for instance N-substituted 6-APA, are remarkablyreduced by contacting the broth, the filtrate of the broth, the eluate,the conversion solution or the dissolved contaminated cephalosporinaccording to formula (I), typically at pH 5 to 7, with carbon dioxide.Carbon dioxide can be added to the solution in any suitable way, such assolid or gaseous form or as solution of carbonate ions. The solution iscontacted with the CO₂ source at a temperature of 10 to 60° C.,preferably 20 to 40° C., where said solution is saturated with molecularCO₂ for 4 to 10 hours. After reduction of the penicillin components,purification of the cephalosporins, according to formula 1 can beobtained as mentioned earlier.

The complex mixture as defined herein may have any origin, but ispreferably a culture broth or a culture filtrate obtained afterfermenting under conditions giving rise to production, a micro-organismcapable of producing an 7-N-acylated version of the compound of thegeneral formula (I), wherein the acyl-group may be any acyl-group whichsupports the ring-expanding enzyme (desacetoxycephalosporinsynthetase—DAOCS—or a bifunctional expandase/hydroxylase occasionallyreferred to as desacetylcephalosporin synthetase DACS) in thecephalosporin biosynthetic pathway. Bioprocesses for producing 7-Nacyl-substituted compounds according to formula (I) in vivo aredisclosed in WO 93/05158 (adipyl-7-ADCA); WO 93/08287 (adipyl-7-ADAC andadipyl-7-ACA), WO 95/04148 (2-(carboxyethylthio)acetyl-7-ADCA), WO95/04149 (3-(carboxyethylthio)propionyl-7-ADCA) and higher alkylsaturated or unsaturated dicarboxylic acids. The relevant parts of thesePCT-applications are herein incorporated by reference. Preferred acylgroups are dicarboxylic acid groups in general, such as adipyl(1,4-dicarboxybutane), 2-(carboxyethylthio)acetyl,3-(carboxyethylthio)propionyl, muconic acid and the like. Suitable hostorganisms include but are not limited to Penicillium chrysogenum andAcremonium chrysogenum. Suitable sources of expandases, includinabifunctional expandase/hydroxylases include but are not limited toStreptomyces clavuligerus and Acremonium chrysogenum. Methods fortransformation, selection of transformed cells and expression regulatingelements for filamentous fungi, which may be used to genetically modifyhost cells, are well known in the art of recombinant DNA technology ofβ-lactam producing (filamentous) fungi.

Preferably, the broth is subjected first to biomass separation such asfiltration by any suitable means, such as membrane filtration, vacuumfiltration, ultrafiltration or a combination thereof, prior toacidification and the optional temperature increase. Any other means ofbiomass separation is suitable as well.

After the pH-lowering step and the optional temperature step, therecovered compound according to the formula (I) is preferably subjectedto further purification to remove, at least partially, unwanted β-lactamcomponents, especially unwanted N-substituted cephalosporins andpenicillins. The further purification may be carried out by extractionusing an organic solvent. In the case of extraction, it is found to beadvantageous to wash the extract, back extract the N-substitutedcephalosporin from the organic phase to an aqueous phase and strippingthe aqueous phase. The extracting organic solvent may be selected fromamyl acetate, butyl acetate, ethyl acetate, methyl isobutyl ketone,cyclohexanone, iso-butanol or n-butanol, and the like. A preferredpurification step in the process is the usage of chromatography for thepurification of N-substituted cephalosporin, rather than extractionusing organic solvents. The advantage of chromatography is in theabsence of solvents, which cause waste problems and problems ofcontainment, as well as improved purity of the final product. Preferredis ion exchange chromatography or adsorption chromatography, morepreferably Hydrophobic Interaction chromatography. The filtrate issubjected to chromatography using an adsorbent. An adsorbent includesactivated carbon, e.g. Norit: CG-1 or Cecarbon GAC 40; or an adsorberresin, such as styrene-divinylbenzene copolymerisates, for exampleDianion HP(CAS No. 55353-13-4), Dianion HP 21 (CAS No. 92529-04-9),Dianion SP 207 (CAS No. 98225-81-1) or Dianion SP825, from MitsubishiKasei Corporation or Amberlite XAD 1180 (CAS No. 97396-56-0), AmberliteXAD 1600 (CAS No. 153796-66-8) or Amberlite XAD 16 (CAS No. 102419-63-8)from Rohm and Haas or Amberchrom CG 161 (CAS No. 131688-63-6) fromTosoHaas; preferably XAD 16 or XAD 1600.

Before adsorbing the N-substituted cephalosporin the complex mixture isadjusted to a pH of 1.0 to 5.0, preferably 2.5 to 3.5, by the means ofone or more known acids, for instance sulphuric acid, hydrochloric acidor nitric acid or a combination thereof. Operating temperature is therange of 0 to 50° C., preferably at 5 to 25° C. Operating pressure is inthe range of 0 to 1.0 MPa overpressure.

Unwanted β-lactam components, especially unwanted N-substitutedcephalosporins, such as alpha-aminoadipylcephalosporanic acids, alsoadsorb on the adsorbent but are displaced by the wanted N-substitutedcephalosporin.

After adsorbing, washing with water is applied to remove unwantedβ-lactam components from the void volume between the adsorbent and todesorb weakly bound unwanted β-lactam components from the adsorbent. Thewater can be acidified down to a pH of 1.0 by the means of one or moreknown acids, for instance sulphuric acid, hydrochloric acid or nitricacid or a combination thereof. To increase the osmotic pressure, saltsmay be added to the water. Operating temperature is the range of 0 to50° C., preferably at 20 to 40° C. Operating pressure is in the range so0 to 1.0 MPa overpressure.

Elution may be carried out with a suitable buffer, such as acetate,phosphate, carbonate, bicarbonate or adipate but also diluted organicsolvents (e.g. acetone, isopropanol) or diluted bases (e.g. ammonium,caustic) can be used. Operating temperature is the range of 0 to 80° C.,preferably at 10 to 40° C. Operating pressure is in the range of 0 to1.0 MPa overpressure.

Regeneration of the adsorbent can be done by any common applied method,such as with dilute bases, dilute acids, or with water miscible solvents(such as acetone, methanol, ethanol or iso-propanol), or a combinationthereof. Optionally heating up to 100° C. may be performed.

The regeneration liquids can be removed by washing with water. The watercan be acidified down to a pH of 1.0 by the means of one or more knownacids, for instance sulphuric acid, hydrochloric acid or nitric acid ora combination thereof.

The chromatography step can be performed in several types of equipment,such as in a single column but also the simulated moving bed technologycan be applied. For this simulated moving bed technology several typesof equipment are available, such as the ADSEP system from U.S. Filter,the ISEP/CSEP-system from Advanced Separation Technology, the‘merry-go-around’-system from e.g. Applexion or the SORBEX-system fromUniversal Oil Products Company (UOP)

Alternatively, the buffer can be removed from the eluate by means ofnanofiltration. The characteristics of the membrane in this membranefiltration show a high retention for the wanted N-substitutedcephalosporin and a low retention for the buffer.

Optionally a concentration step is applied by any means of suitableconcentration such as vacuum evaporation, reversed osmosis,nanofiltration, or nanofiltration after chromatography or extraction.

The recovered N-acylated compound is subsequently subjected todeacylation using any suitable method Known in the art. A Preferredmethod is enzymatic deacylation using a suitable dicarboxylate acylase.Numerous suitable acylases, wild-type or mutated, are known in the artincluding but not limited to those from Bacillus (EP 0 525 861; EP 0 405846), Pseudomonas (EP 0 482 844; EP 0 525 861; EP 0 475 652; EP 0663445), Achromobacter (EP 0 525 861), Alcaligenes faecalis (EP 0 638 649),Acinetobacter (EP 0 469 919), Arthzrobacter (EP 0 283 218), Escherichiacoli (U.S. Pat. No. 3,945,888), Kluyvera citrophila, Proteus rettgeri(U.S. Pat. No. 3,915,798) and the like. The dicarboxylate acylase ispreferably from Pseudomonas SE83 or SY-77. Optionally, the acylase maybe a mutated form, as disclosed in WO 91/16435, WO 97/20053, WO 97/40175to increase or alter the affinity towards the substrate. Another way ofdeacylating the N-acylated cephalosporin compound according to theinvention is by way of contacting the substrate with a micro-organismcapable of producing the acylase, as disclosed in U.S. Pat. No.5,677,141.

The acylase may be immobilised (U.S. Pat. No. 3,930,949), either onmembranes (EP 0 243 404) or free flowing carriers such as glutaraldehydebased carriers or aza-lacton polymers (EP 0730 035), using technigues assuch well known in the art. Non-immobilised acylase is alsocontemplated, using membranes to separate the reaction mixture(retentate) from the product (permeate), such as disclosed in U.S. Pat.No. 5,521,068. The process may be batch-wise or (semi-)continuous, thisis all well known and not crucial with respect to the invention. Theenzymatic deacylation reaction is usually carried out in a stirred tankreactor with or without, preferably inert, sieve plates, to easilyseparate the immobilised enzyme from the reaction product. The pH isusually regulated during the reaction to compensate for the pH change asa result of the (dicarboxylic) side-chain removal by any type of basesuch as ammonium, caustic, carbonate, bicarbonate. The pH can beregulated in the reactor and/or in a circulating loop over the reactor.Other parameters may also be regulated, such as temperature, deacylatedproduct or side-chain concentration, and the like, taking account of theeffect of such Parameters on the reaction rate and/or the equilibrium.

Additional stabilising agents can be added before and/or duringdeacylation, such as sulphite (S₂O₅ ²⁻, HSO₃ ⁻, SO₃ ²⁻), EDTA,dithiotreitol (DTT)

Usually, the deacylated cephalosporin compound of the general formula(I) is subsequently recovered using any suitable combination of steps.Optionally a concentration step can be applied by any means such asvacuum evaporation, reversed osmosis, nanofiltration, or narofiltrationbefore crystallisation. Optionally a water miscible solvent can beadded. Optionally, before crystallisation the solution can be purifiedby treating with activated carbon or an adsorber resin. Optionally,before crystallisation the side chain can be removed, characterised byacidifying the aqueous phase, extracting the side chain to an extractingorganic solvent and separating the phases. The extracting organicsolvent may be selected from amyl acetate, butyl acetate, ethyl acetate,methyl isobutyl ketone, cyclohexanone, iso-butanol, n-butanol, and thelike.

The product can be crystallised from the resulting aqueous phase inseveral ways. The most preferred mode of operation is neutralising theaqueous solution and subsequently lowering the pH in 1 to 6 steps downto a pH 3 to 5 using one or more known acids such as H₂SO₄, HCl, HNO₃,or a combination thereof. This is preferably carried out in continuousmode using an interconnected set of 1 to 6 continuously operatedcrystallisers in series. Also batch crystallisation, semi-continuouscrystallisation or concordance crystallisation can be applied. It ispossible to perform the crystallisation directly in the same way asabove, without the first neutralisation. According to one embodiment ofthis invention it has been found that a water miscible solvent, such asmethanol, ethanol, iso-propanol, n-butanol, acetone and the like, can beadded to improve the quality of the cephalosporin according to formula(II). Optionally, before crystallisation the solution can be treated byactivated carbon or by an adsorbent resin in order to improve thequality of the compound according to formula (II).

It has been found that the quality of the cephalosporin according toformula (II) can be further improved by recrystallisation, optionallyafter treatment with adsorber resins, active coal and/or ethanol and/oracetate. This is characterised by dissolving the cephalosporin accordingto formula (II) at a pH in the range of 0.5 to 10.0, preferably between7.5 to 8.5 and crystallisation of the product. The product can becrystallised in several ways. The most preferred mode of operation islowering of the pH in 1 to 6 steps down to a pH 3 to 5 using one or moreknown acids, such as H₂SO₄, HCl, HNO₃, or a combination thereof. Thiscan be carried out in continuous mode using an interconnected set of 1to 6 continuously operated crystallisers in series. Also batchcrystallisation, semi-continuous crystallisation or concordancecrystallisation can be applied. According to one embodiment of thisinvention it has been found that a water miscible solvent, such asmethanol, ethanol, (iso)propanol, acetone, iso-butanol and n-butanol,can be added to improve the quality of the cephalosporin according toformula (II).

It has been found further, that the quality of the cephalosporinaccording to formula (II) can be improved by treating the conversionsolution and/or the solution of the dissolved cephalosporin according toformula (II) with an adsorbent. An adsorbent includes activated carbon,e.g. Norit Ultra SX; or an adsorber resin, such asstyrene-divinylbenzene copolymersates, for example Dianion HP 20 (CASNo. 55353-13-4), Dianion HP 21 (CAS No. 92529-04-9) or Dianion SP 207(CAS No. 98225-81-1) from Mitsubishi Kasei Corporation or Amberlite XAD1180 (CAS No. 97396-56-0), Amberlite XAD 1600 (CAS No. 153796-66-8) orAmberlite XA 16 (CAS No. 102419-63-8) from Rohm and Haas or AmberchromCG 161 (CAS No. 131688-63-6) from TosoHaas; preferably XAD 16, XAD 1600or HP20.

The crystals are isolated by filtration or centrifugation and dried in aconventional continuous or batch dryer. The crystals can be milled byany type of mill, such as ball mill, jet mill and the like.

Optionally a water miscible solvent can be added during thecrystallisation. After dissolving, the solution can be treated withactivated carbon or an adsorber resin.

This procedure will gave a better overall yield and product quality thanthe currently known process, mentioned before.

According to another aspect of the invention, a method is provided forremoving and recovering adipic acid from the conversion solution ormother liquid (the liquid obtained after crystallisation of the compoundaccording to the formula (II)). It is found, that adipic acid canadvantageously separated using membrane filtration at low pH, such asbelow pH 5, preferably below pH 4, more preferably below at or near pH3. Preferred according to the invention is an embodiment whereinfiltration is carried out by reversed osmosis.

In addition to saving raw materials, the advantage of doing so residesin the purity and/or yield upon crystallisation of the so-treatedsolution.

The invention is further illustrated by the following non-limitingexamples.

EXPERIMENTAL

A fermentation broth comprising adipyl-7-ADCA as a complex mixture,comprising inter alia 6-APA, adipyl-6-APA andalpha-amino-adipyl-7-cephalosporanic acid as undesired contaminants, isobtained by fermenting a Penicillium chrysogenum strain transformed withan expandase (desacetoxycephalosporin C synthetase) from Streptomycesclavuligerus, as described in International patent application WO93/05158, published so Mar. 18, 1993.

The transformed Penicillium strain was cultured as described in Example1 of WO 93/05158, incorporated by reference herein.

After 5 to 7 days of fermentation, he broth was taken for recoveryexperiments.

This complex mixture can also be simulated by making an aqueous mixtureof 6-aminopenicillanic acid, adipyl-6-aminopenicillanic acid,alpha-aminoadipyl-6-amino-penicillanic acid,adipyl-7-aminodesacetoxycephalosporanic acid, andalpha-aminoadipyl-7-cephalosporanic acid.

EXAMPLE 1 pH/Heat-treatment

This example shows the advantages of a pH-treatment, preferably acombined pH-plus increased temperature treatment, on the removal ofunwanted β-lactam components, from complex mixtures.

Broth from a fermentation of Penicillium chrysogenum (see experimental),containing a complex mixture of adipyl-7-ADCA and penicillanic acid andcephalosporanic acid contaminants is filtrated. The concentrate iswashed with process water until the total volume of the combinedfiltrates was approx. 2 times the initial broth volume. The followingexperiments have been carried out:

A. Part of the filtrate is acidified to pH=3.5 heated up to 70° C. andafter 30 minutes cooled to 4° C.;

B. Part of the permeate is acidified to pH=2.7; heated up to 110° C. andafter 4 minutes cooled to 25° C.; or

C. Part of the permeate is acidified to pH=3.0 and not further treated.

The pre-treated solutions are then subjected to the following treatmentsto obtain a compound according to formula (II); 7-ADCA.

Adsorption Chromatography

The three solutions (A to C) were subjected to filtration over a SeitzK100 filter, whereafter the solution was pumped at a pH of 3.0 over acolumn filled with 1.6 liter of XAD-1600 resin; next the resin waswashed with 4.8 liter water, and eluted with 0.2 M bicarbonate-solution.The first eluate fraction (1.1 liter) is taken out and discarded. Thesecond fraction (3.2 liter) is collected and analysed. The resin ispurified by washing with caustic and acetone, and conditioned again withacidified water.

Concentrating

The eluate was concentrated at 20 to 30° C. vacuum (5-10 mm Hg) till aconcentration of 40 grams adipyl-7-ADCA per liter was obtained.

Enzymatic Deacylation

Subsequently, the adipyl-7-ADCA is treated with acylase as follows. To 1liter of eluate, 1 gram of sodium metabisulfite, 20 mM EDTA and 100 gimmobilised acylase (comprising Pseudomonas SE83 dicarboxylate acylase)was added. At 30° C., the solution was stirred for two hours. The pH washeld at 8.5 with 4 N sodium hydroxide. The immobilised acylase and theliquid were separated with a glass sintered filter.

Crystallisation of 7-ADCA

The 7-amino desacetoxy cephalosporanic acid (7-ADCA) was precipitated bylowering the pH to 3.6, under stirring, at a temperature of 30° C.; in45 minutes the pH of the solution was lowered to 3.6 with 6 N sulphuricacid. After cooling to 20° C., the crystals were isolated on a glasssintered filter, washed with water and dried at 35° C.

Resolving 7-ADCA Crystals

The 7-ADCA was dissolved with the aid of ammonia. To that end 15 gramsof 7-ADCA was suspended in 255 ml water. The 7-ADCA was dissolved withthe aid of 4 N ammonium hydroxide at a pH of 7.5-8.5. After filtrationover a glass sintered filter, water was added to obtain 300 ml ofsolution.

Treatment with Adsorber Resin

The solution was treated with adsorber resin. In 45 minutes the solutionwas pumped over 15 ml of XAD1600. Subsequently, 75 ml of water waspumped over the resin to obtain 375 ml of solution.

Recrystallisation

The 7-ADCA was precipitated by lowering the pH to 3.6 under stirring, ata temperature of 30° C.; in 45 minutes the pH was lowered to 3.6 with 6N sulphuric acid. After cooling to 20° C., the crystals were isolated ona glass sintered filter, washed with water and dried at 35° C.

The 7-ADCA so produced shows good results in terms of 6-APA reduction.(6-APA ratio is with respect to 7-ADCA).

TABLE 1a Results of experiment 1A, 1B and 1C. 6-amino penicillanic acidcontent Experiment (ppm) 1A <10 1B <10 1C 950

Clearly, the pH/temperature treatment reduces the level of6-aminocenicillanic acid contamination of the adipyl-7-ADCA preparation.

The relationship between pH, Temperature and Time of treatment wasdetermined for a fixed reduction of 6-aminopenicillanic acid of 10⁻⁶(Table 1b).

TABLE 1b 6-APA Temp. Time Time Time reduction pH (C.) (s) (min) (h) 10⁻⁶3 25 35050 584 9.74 10⁻⁶ 3 50 3057 50.9 0.85 10⁻⁶ 3 75 378 6.3 0.11 10⁻⁶3 100 62 1.0 0.02 10⁻⁶ 4 25 148857 2481 41.35 10⁻⁶ 4 50 12982 216.4 3.6110⁻⁶ 4 75 1607 26.8 0.45 10⁻⁶ 4 100 263 4.4 0.07

EXAMPLE 2 Adsorption Chromatography

This example shows the effect of (a) the degree of loading of the columnwhen adsorption chromatography is used (2A to 2D), (b) the effect ofwashing the column with different amounts of water prior to elution (2Eto 2G), (c) the effect of the pH of the feed on the purification ofadipyl-7-ADCA (2F to 2J). The embodiment where adsorption chromatographyis carried out in a Simulated Moving Bed mode is given as Experiment 2K.

The broth is pre-treated as described in Example 1A. Theadipyl-7-amino-desacetoxy cephalosporanic acid was subsequently purifiedby adsorption chromatography by pumping the solution over a columnfilled with 1.6 liter of XAD-1600 resin, washed with different amountsof water (2A to 2D and 2H to 2K: 4.8 liter; 2E to 2F: see Table 2b), andeluted with 0.2 M bicarbonate-solution. The first eluate fraction (1.1liter) is taken out and discarded. The second fraction (3.2 liter) iscollected and analysed. The resin is purified by washing with causticand acetone, and conditioned again with acidified water. Several changesin process conditions were applied (see table 2). Reduction iscalculated as:(comp-i_(feed)/comp-1_(feed))/(comp-i_(eluate)/comp-1_(eluate)).

TABLE 2a Results of experiment 2 Feed Eluate (g) Reduction comp compcomp comp comp comp (−) comp 1 2 3 1 2 3 comp comp 4 Exp (g) (g) (g) (g)(g) (g) 2 3 (ppm) 2A 34 3.3 9.3 30 3.1 5.9 1 1 2B 80 5.9 18.5 70 0.50.09 10 173 <6 2C 127 10.5 32.8 76 0.3 0.07 24 301 19 2D 255 18.2 59.467 0.1 0.03 38 501 30 comp 1: adipyl-7-ADCA comp 2: alpha-hydroxyadipyl-7-ADCA comp 3: alpha-amino adipyl-7-ADCA comp 4: 6-APA contentrelative to comp 1

These results clearly show the positive effect of overloading the columnon the reduction of compounds 2 and 3 in the eluate.

TABLE 2b Results of experiment 2 Feed Eluate Reduction comp comp compcomp comp comp (−) 1 2 3 Wash 1 2 3 comp comp Exp (g) (g) (g) (1) (g)(g) (g) 2 3 2E 85 6.6 14.0 1.6 81 2.1 1.2 3 11 2F 74 6.4 12.6 4.8 71 1.10.13 6 94 2G 75 5.8 13.0 7.3 68 0.5 0.1 12 122 comp 1: adipyl-7-ADCAcomp 2: alpha-hydroxyadipyl-7-ADCA comp 3: alpha-amino adipyl-7-ADCA

The example 2b shows the positive effect of extended washing, prior toelution with sodium bicarbonate, on the reduction of undesiredcephalosporin compounds.

TABLE 2c Feed Eluate Reduction comp comp comp comp comp comp (−) 1 2 3pH 1 2 3 comp comp Exp (g) (g) (g) (−) (g) (g) (g) 2 3 2H 79 8.4 22.22.5 83 0.6 0.09 15 248 2I 80 5.9 18.5 2.9 70 0.5 0.09 10 183 2J 83 8.221.7 3.5 62 0.5 0.14 12 118 comp 1: adipyl-7-ADCA comp 2: alpha-hydroxyadipyl-7-ADCA comp 3: alpha-amino adipyl-7-ADCA

The above example shows the effect of the pH at which crystallisationwas carried out, on the reduction of unwanted 7-N acylated cephalosporincompounds. H₂SO₄ was used as acid.

TABLE 2d Results of experiment 2 (30 liters of resin in an SMB-systemwas applied) Feed Eluate comp comp comp comp comp comp comp compReduction (−) 1 2 3 4 1 2 3 4 comp comp comp Exp (kg) (kg) (kg) (kg)(kg) (kg) (kg) (kg) 2 3 4 2K 1.46 0.08 0.20 0.15 1.38 0.01 0.01 0.02 718 7 comp 1: dipyl-7-ADCA comp 2: alpha-hydroxyadipyl-7-ADCA comp 3:alpha-aminoadipyl-7-ADCA comp 4: adipic acid

This example illustrates the use of adsorption chromatography performedaccording to the so-called Simulated Moving Bed technique, on a kilogramscale. The technique may readily be scaled up further.

The so treated fractions 2A to 2K were treated with acylase to produce7-ADCA as described in Example 1. Excellent conversion results wereobtained, as illustrated in Example 3.

EXAMPLE 3 Enzymatic Conversion

This example illustrates the results of enzymatic conversion ofadipyl-7-ADCA to 7-ADCA. The adipyl-7-ADCA was recovered as disclosed inExample 2K (pH-treatment according to Example 1A, adsorptionchromatography was optimised in terms of overloading and washing). Theconversion was carried out as described in Example 1, at the pHindicated in Table 3. Experiment A to E represent different batches.

TABLE 3 Product product substrate Substrate stream stream comp 1 comp 2pH comp 1 comp 2 Exp (mmol) (mmol) (−) (mmol) (mmol) A 69.7 2.2 8.5 1.168.6 B 144.2 2.4 8.5 5.9 143.3 C 181.4 2.3 8.5 13.5 174.5 D 113.1 1.7 85.4 108.7 E 113.4 3.1 9 1.2 112.2 comp 1: adipyl-7-ADCA comp 2: 7-ADCA

The conversion rate and yields are superior when the adipyl-7-ADCA ispre-treated using the pH/temperature step, as compared to no treatment.The further purification using chromatography brings further improvementin terms of purity (not shown in the Table).

EXAMPLE 4 Crude Crystallisation

The broth is pH/heat-treated (Example 1) and enriched in adipyl-7-ADCAby adsorption chromatography as described in Example 2. Subsequently,conversion was carried out as described in Example 1.

The conversion solution (the solution obtained after deacylation) wasconcentrated with reversed osmosis to increase the concentration.

Part of the solution was taken and the 7-ADCA was crystallised bylowering the pH to the pH 3.6, 4 or 5 (see table 4a).

TABLE 4a Crude crystallisation product after comp 1 comp 1 isolation, inin pH washing and product Exp solution (−) drying (g) (%) A 49.5 3.648.9 97.5 B 49.5 4 48.7 97.4 C 49.5 5 48.2 98 comp 1: 7-ADCA

Crystallisation was satisfactory at all pH tested. In the followingexperiment the pH was 3.6. The effect of concentrating the solution isillustrated.

TABLE 4b Crude crystallisation product after comp 1 comp 1 isolation, inin washing and product Exp solution (g) drying (g) (%) D 15.5 14.8 94.3E 36.1 35.7 95.3 F 49.5 48.9 97.5 comp 1: 7-ADCA

Clearly, there is an effect of the concentration of 7-ADCA in theconversion solution on purity and yield after crystallisation.

The following example illustrates the effect of different adsorbers onproduct quality (colour in solution and clarity).

TABLE 4c Crude crystallisation colour in comp 1 comp 1 solution clarityin in at 425 in HCl Exp solution (g) treatment product (%) nm (−) (EBC)G 25 HP20 97.2 0.16 3.6 H 25 HP20 97.8 0.11 2.4 (2 times) I 25 1RA6797.2 0.18 6 J 25 IRA67 + HP20 97.7 0.1 0.8 K 25 none 96.3 0.39 7.3 comp1: 7-ADCA

EXAMPLE 5 Treatment of Dissolved 7-Amino Desacetoxy Cephalosporanic Acid

This example shows the effect on clarity and colour of 7-ADCA, aftertreating 7-ADCA solution with different adsorber resins, prior tocrystallisation.

A solution comprising 7-ADCA is made as disclosed in Example 2K (theadsorption chromatography column used is a XAD-1600 resin).

TABLE 5 Colour in Clarity comp 1 solution in HCl Exp dissolved (g)treatment at 425 nm (−) (EBC) A 40 — 0.18 4.2 B 40 XAD16 0.05 1.8 C 40HP20 n.d. 0.5 D 40 XAD1600 0.09 0.5 E 25 n.d. 3.6 F 25 +1% EtOH 0.11 0.6G 50 +3% EtOH 0.18 0.9 H 50 +2% Coal 0.04 n.d. I 50 0.17 1.4 J 50 +5%Coal 0.03 0.8 comp 1: 7-amino desacetoxy cephalosporanic acid

EXAMPLE 6 Recovery of Adipyl-7-ADCA Using Extraction with N-Butanol

Broth comprising adipyl-7-ADCA is treated as described in Example 1.

After acidification, part of the adipyl-ADCA is purified by adsorptionchromatography. The solution is pumped over a column filled with XAD-16resin, washed with water, and eluted with 0.2 M acetate-solution. Thefirst eluate fraction with low adipyl desacetoxy cephalosporanic acidcontent is taken out and discarded. The second fraction is collected.The resin is purified by washing with caustic and acetone, andconditioned again with acidified water.

Part of the adipyl-7-ADCA is purified by means of extraction, followedby washing of the extract, back extraction of the N-substitutedcephalosporin from the organic phase to an aqueous phase and strippingthe aqueous phase; the extracting organic solvent is n-butanol.

The adipyl-7-ADCA is treated with immobilised acylase to produce 7-aminodesacetoxy cephalosporanic acid (7-ADCA). Part of the 7-ADCA is isolatedby lowering the pH. The adipyl desacetoxy cephalosporanic acid wasdissolved with the aid of caustic. The 7-amino desacetoxycephalosporanic acid was isolated by lowering the pH.

Part of the 7-amino desacetoxy cephalosporanic acid aqueous solution isacidified, and the side chain is extracted to an extracting organicsolvent and next the phases are separated; the extracting organicsolvent was n-butanol,

Finally the crystal cake was filtrated, washed and dried.

TABLE 6 colour 1 comp 1 in in solution clarity product at 425 nm in HClExp description (%) (−) (EBC) A extraction/extraction/ 98.3 0.13 2.4crystallisation B chromatography/extraction/ 98.9 0.09 1.5crystallisation C chromatography/ 97.6 0.12 — crystallisation Dchromatography/ 98.7 0.05 1.1 crystallisation/ recrystallisation Comp 1:7-amino desacetoxy cephalosporanic acid

The results using a single extraction are not shown in the table, butthe purity is far worse than when chromatography is used, even withoutrecrystallisation. A combination of chromatography and recrystallisationproduces or a combination of chromatography and extraction produces thebest results.

EXAMPLE 7 Recovery of Adipic Acid from 7-ADCA Crystallisation MotherLiquors

7-ADCA crystallisation mother liquors are obtained as described inexample 4. Adipic acid is determined using HPLC: Aminex HPX-87H column,300 mm×7.8 mm, filled with 9 um cation gel (Biorad), eluted at 65° C.with a 0.2M solution of H₂SO₄ in water, detection using an RI Waters 410refractometer. In the examples given below, optimisation is directed atpurity, not at yield.

EXAMPLE 7A Recovery of Adipic Acid Using Acidification

At 20° C., the pH of 7-ADCA crystallisation mother liquor (250 ml, 13.6g/l adipic acid) was lowered to 0.7 using a 12M solution of H₂SO₄ inwater. After 16 h at 0° C., no crystallisation could be detected. The pHwas raised to 3.4 using a 6M solution of KOH in water. The resultingcrystal was recovered by filtration to give 7.7 g of material which wasa mixture of salt and adipic acid which was not further analysed.

EXAMPLE 7B Recovery of Adipic Acid Using Acidification and Concentration

At 20° C., the pH of 7-ADCA crystallisation mother liquor (500 ml, 9.4g/l adipic acid) was lowered to 1.5 using a 12M solution of H₂SO₄ inwater and concentrated under reduced pressure at 40° C. to give a stickymixture that was isolated by filtration and dried to give 6.9 g adipicacid with a purity of 53% (yield 78%).

EXAMPLE 7C Recovery of Adipic Acid Using Reverse Osmosis with Nanomax 50Membrane

At 20° C., the pH of 7-ADCA crystallisation mother liquor (500 ml, 9.4to 18.2 g/l adipic acid, see table) was adjusted to the value mentionedin the table using either a 6M solution of KOH in water or a 12Msolution of H₂SO₄ in water. The resulting solution was subjected toreverse osmosis using a Nanomax 50 membrane from Millipore. With the aidof nitrogen gas, a pressure of 30 bar was applied to give a filtrate anda retentate in which the amount of adipic acid was determined usingHPLC. In most cases, a work-up procedure was applied that consisted ofconcentration under reduced pressure until crystallisation began,followed filtration of the product and drying.

TABLE 7 Yield Purity Adipic acid (g/l) Volume after after Per- Retentionpermeate work-up work-up pH Start meate Retentate (%) (ml) (g) (%) 1.513.6 11.0 14.8 26 400 4.4 96 2.0 18.2 13.1 20.8 37 260 2.4 99 3.0 9.46.7 8.7 23 360 1.9 97 7.2 13.6 5.3 31.9 83 400 no no work-up work-up

EXAMPLE 7D Recovery of Adipic Acid Using Reverse Osmosis with DK U19FMembrane at pH 2.0

The pH of 7-ADCA crystallisation mother liquor (100, containing 25.0 g/ladipic acid) was lowered to 2.0 using 2.9 l of a 12M solution of H₂SO₄in water. The resulting solution was subjected to reverse osmosis with57 l water using a DK U19F membrane in a 2.5 m² membrane filtration unitP2-B200 from Hydro Air Research. At a pressure of 30bar an average luxof 8.8 l/m²/h was reached to give the results summarised in the table.

TABLE 8 Concentration (g/l) Retention Component Start Permeate Retentate(%) Adipic acid 25.0 11.6 17.8 35 7-ADCA 0.72 <0.01 0.72 >99 Adipyl-7-1.51 <0.03 1.54 >98 ADCA

What is claimed is:
 1. A method for the recovery from a complex mixtureof a compound of formula (I):

R₀ is hydrogen or C₁₋₃ alkoxy; Y is CH₂, oxygen, sulphur, or an oxidisedform of sulphur; R₁ is selected from the group consisting of hydrogen,hydroxy, halogen, C₁₋₃ alkoxy, R₂ is selected from the group consistingof adipyl, succinct, 2-(carboxyethylthio)acetyl,3-(carboxyethylthio)propionyl, higher alkyl saturated dicarboxylic acidsand higher alkyl unsaturated dicarboxylic acids, wherein said mixturecomprises in addition to the compound of formula (I),6-aminopenicillanic acid (6-APA) and optionally one or more additionalN-substituted β-lactam compounds, comprising the steps of: (a)acidifying the complex mixture to a pH below 6.5 and maintaining themixture below said pH at a temperature of between 50° C. and 130° C.;and/or (b) contacting the complex mixture with a carbon dioxide source;and (c) subjecting the mixture obtained after steps (a) and/or (b) tochromatography to obtain the compound of formula (I).
 2. A methodaccording to claim 1, wherein in step (a) the temperature is keptbetween 70 and 120° C., for between 10 seconds and about 1 day and thepH is kept at or below pH 4.5.
 3. A method according to claim 1, whereinthe compound of formula (1) has been produced by fermentation of amicroorganism capable thereof and wherein the complex mixture is abroth, a culture filtrate or any culture liquid derivable from the brothafter fermentation.
 4. A method for the recovery from a complex mixtureof a compound selected from the group consisting ofadipyl-7-aminodesacetoxycephalosporanic acid (adipyl-7-ADCA),adipyl-7-aminodeacetylcephalosporanic acid (adipyl-7-ADAC) andadipyl-7-aminocephalosporanic acid (adipyl-7-ACA), wherein said mixturecomprises in addition to said compound, 6-aminopenicillanic acid (6-APA)and optionally one or more additional N-substituted β-lactam compounds,comprising the steps of: (a) acidifying the complex mixture to a pHbelow 6.5 and maintaining the mixture below said pH at a temperature ofbetween 50° C. and 130° C.; and/or (b) contacting the complex mixturewith a carbon dioxide source; and (c) subjecting the mixture obtainedafter steps (a) and/or (b) to chromatography to obtain said compound. 5.The method according to claim 1, wherein said chromatography ishydrophobic interaction chromatography.
 6. A method according to claim1, wherein chromatography is adsorption chromatography.
 7. A process forpreparing a compound of formula (II):

comprising deacylating the compound of formula (I) obtained according tothe method of any one of claims 1-3 and 6 to obtain a conversionsolution which comprises a compound according to formula (II).
 8. Theprocess of claim 7, wherein the deacylating comprises treating thecompound of formula (1) with a dicarboxyl acylase.
 9. A processaccording to claim 7, comprising the further step of recovering thecompound of formula (II) from the solution by crystallisation.
 10. Aprocess according to claim 9, wherein crystallisation is preceded bytreating the solution with an agent selected from the group consistingof an adsorber resin, active coal, methanol, ethanol, isopropanol,isobutanol, n-butanol, acetone or a combination of any of the mentionedagents.
 11. A process according to claim 10, wherein the adsorber resincomprises styrene-divinylbenzene copolymerisates.
 12. A method accordingto claim 7, wherein the 6-aminopenicillanic acid (6-APA) level is 10 ppmor less with respect to the compound of formula (II).
 13. A processaccording to claim 9, which comprises the further step of removing, atleast partially, the cleaved side chain R₂OH.
 14. A process according toclaim 13, wherein said removing is carried out on the mother liquorobtained after crystallisation.
 15. A process according to claim 14,wherein said removing is followed by solubilizing and recrystallizingthe compound of formula (II).
 16. A process according to claim 14,wherein crystallisation is preceded by treating the solution with anagent selected from the group consisting of an adsorber resin, activecoal, methanol, ethanol, isopropanol, isobutanol, n-butanol and acetone,or a combination of any of these mentioned agents.
 17. A processaccording to claim 13, wherein said removing comprises subjecting theconversion solution, or the mother liquid, to membrane filtration at apH below 5.